The superfamily of G-protein coupled receptors (GPCRs) represents the largest family of cell surface receptors, and is one of the most important sources of drug targets for the pharmaceutical industry. GPCRs are involved in a wide range of disorders and disease states including ulcers, psychosis, anxiety, Parkinson's disease, Alzheimer's disease and hypertension. More than 20% of the bestselling prescription drugs and an estimated 50% of all prescription drugs interact directly with a GPCR. Also, interactions of drugs with this class of receptors are responsible for some of the side effects associated with these drugs.
Over 500 different GPCRs have been identified in the human genome. As many as 200 of these represent ‘orphan’ receptors, for which the natural ligand is unknown. The first step for the transformation of orphan receptors into drug targets is their characterization, or ‘de-orphanization’ (AD Howard et al., 2001, Orphan G-protein-coupled receptors and natural ligand discovery, Trends in Pharmacol. Sci. 22(3): 132-140). De-orphanization of GPCRs, and the identification of synthetic agonists and antagonists, will likely lead to a large number of new and potent medicines for various conditions. This is a task that involves significant efforts, both to understand the potential importance of receptor candidates to specific diseases and to develop efficient drug screening tools. For example, ligands of an identified receptor can be tested against related orphan GPCRs to identify compounds that bind to the orphan receptor. In addition, extracts of tissues can be tested using functional assays to guide ligand fractionation, purification and molecular characterization. Finally, orphan GPCRs can be evaluated against arrayed families of known ligands. Sensitive, biologically relevant assays are needed that can be used to de-orphanize GPCRs on a large scale.
GPCRs do not share any overall sequence homology, but have in common the presence of seven transmembrane-spanning alpha-helical segments connected by alternating intracellular and extracellular loops, with the amino terminus on the extracellular side and the carboxyl terminus on the intracellular side of the cell membrane. Therefore, GPCRs are commonly referred to as seven-transmembrane (7TM) receptors. The GPCRs have been divided into different subfamilies (A-F); the major subfamilies, A, B and C, include the beta-2-adrenergic receptor (family A), receptors related to the glucagon receptor (family B) and receptors related to the neurotransmitter receptors (family C). Family B includes the receptors for vasoactive intestinal peptide, calcitonin, PTH and glucagon; family C includes the receptors for GABA, calcium, mammalian pheromones, and taste receptors. All GPCRs signal through guanine nucleotide-binding proteins (G-proteins). The DNA sequences of a large number of GPCRs can be found in public databases, among other sources (F. Horn et al., 1998, GPCRDB: an Information system for G protein-coupled receptors, Nucleic Acids Res. 26:275-279). The public GPCR database can be found on the worldwide web and corresponding cDNAs and pairwise sequence alignments can also be found on the worldwide web. This database is incorporated herein by reference.
The general mechanism of action of GPCRs in cell signaling has been elucidated over the last 20 years, although many details remain to be discovered. Hundreds of scientific and review articles have been written on the topic (for reviews see GB Downes & N Gautham, 1999, The G-protein subunit gene families, Genomics 62: 544-552; Hermans, 2003, Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors, in: Pharmacology & Therapeutics 99: 25-44; and U Gether, 2000, Uncovering molecular mechanisms involved in activation of G-protein-coupled receptors, in: Endocrine Reviews 21: 90-113; E M Hur & K T Kim, 2002, G-protein-coupled receptor signaling and cross talk: achieving rapidity and specificity, Cell Signal 14: 397-405). Some of the known elements of the various G-protein-coupled pathways are described herein for the purposes of placing the present invention in the context of the prior art. Further elucidation of the signaling pathways linked to GPCRs would allow the construction of a large number of assays for intracellular events linked to GPCR activation. In turn, such assays would allow drug discovery to identify drug candidates capable of activating or blocking GPCR signaling.
For drug discovery, there is a need to quickly and inexpensively screen large numbers of chemical compounds to identify new drug candidates, including agonists, antagonists and inhibitors of GPCRs and GPCR-dependent pathways. These chemical compounds are collected in large libraries, sometimes exceeding one million distinct compounds. The use of the term chemical compound is intended to be interpreted broadly so as to include, but not be limited to, simple organic and inorganic molecules, proteins, peptides, antibodies, nucleic acids and oligonucleotides, carbohydrates, lipids, or any chemical structure of biological interest. Traditional, biochemical approaches to assaying GPCRs have relied upon measurements of ligand binding, for example with scintillation proximity assays or with surface plasmon resonance (C. Bieri et al., 1999, Micropatterned immobilization of a G-protein-coupled receptor and direct detection of G protein activation, Nature Biotech. 17: 1105-1109). Although such assays are inexpensive to perform, they can take 6 months or longer to develop. A major problem is that the development of an in vitro assay requires specific reagents for every target of interest, including purified protein for the target against which the screen is to be run. Often it is difficult to express the protein of interest and/or to obtain a sufficient quantity of the protein in pure form. Moreover, although in vitro assays are the gold standard for pharmacology and studies of structure activity relationships (SAR) it is not possible to perform target validation with an in vitro assay, in vivo assays are necessary in order to obtain information about the biological availability and cellular activity of the screening hit.
The increased numbers of drug targets identified by genomics approaches has driven the development of ‘gene to screen’ approaches to interrogate poorly defined targets, many of which rely on cellular assay systems. Speculative targets are most easily screened in a format in which the target is expressed and regulated in the biological context of a cell, in which all of the necessary components are pre-assembled and regulated. Cell-based assays are also critical for assessing the mechanism of action of new biological targets and the biological activity of chemical compounds. In particular, there is a need to ‘de-orphanize’ those GPCRs for which the natural activating ligand has not been identified. Various approaches to de-orphanization have been reviewed (A D Howard et al., 2001, Orphan G-protein-coupled receptors and natural ligand discovery, Trends in Pharmacological Sciences 22: 132-140). For example, extracts of tissues can be tested using functional assays to guide ligand fractionation, purification and molecular characterization. Alternatively, orphan GPCRs can be evaluated against arrayed families of known ligands.
Current cell-based assays for GPCRs include measures of pathway activation (Ca2+ release, cAMP generation, or transcriptional activity); measurements of protein trafficking by tagging GPCRs and downstream elements with GFP; and direct measures of interactions between proteins using fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET) or yeast two-hybrid approaches (e.g. King et al., U.S. Patent Application 20020022238). These approaches are described below.
The majority of cell-based assays for GPCRs rely upon measurements of intracellular calcium. Calcium release from intracellular stores is stimulated by specific classes of GPCRs upon their activation; in particular, those GPCRs that couple to Gq. Fluorescent and luminescent assays of calcium release have been generated by loading cells with dyes that act as calcium indicators. Fluorescent Ca2+ indicators such as fura-2, indo-1, fluo-3, and Calcium-Green have been the mainstay of intracellular Ca2+ measurement and imaging (see for example U.S. Pat. Nos. 4,603,209 and 5,049,673). Such indicators and associated instrumentation systems (FLIPR system) are sold, for example, by Molecular Devices of Sunnyvale Calif. (www.moleculardevices.com). Luminescent assays of calcium flux can be accomplished by introducing aequorin into cells. Aequorin emits blue light in the presence of calcium, and the rate of photon emission is proportional to the free Ca2+ concentration within a specific range. Cells expressing the GPCR of interest are loaded first with coelenterazine to activate the aequorin, and then the compounds to be tested are added to the cells and the results quantitated with a luminometer. To extend these assays to non-Gq-coupled receptors, various strategies have been employed, including the use of a promiscuous Gα protein such as Gα16 that is capable of coupling a wide range of GPCRs to phospholipase C (PLC) activity and calcium mobilization (Milligan et al., 1996, Trends in Pharmacological Sciences 17: 235-237).
Fluorescent dyes, and fluorescent proteins such as GFP, YFP, BFP and CFP, have also been used as cellular sensors of cAMP or Ca2+. The first fluorescent protein indicator for cAMP consisted of the cyclic AMP-dependent protein kinase, PKA, in which the catalytic and regulatory subunits were labelled with fluorescein and rhodamine, respectively, so that cAMP-induced dissociation of the subunits disrupted FRET (S. R. Adams et al., 1991, Fluorescence ratio imaging of cyclic AMP in single cells, Nature 349: 694-697). Replacement of the dyes by GFP and BFP made this system genetically encodable and eliminated the need for in vitro dye conjugation and microinjection (M. Zaccolo et al., 2000, A genetically encoded fluorescent indicator for cyclic AMP in living cells, Nature Cell Biol. 2: 25-29). A variety of other GFP-based techniques have been used to create cellular sensors. For example, two GFP molecules joined by the kinase inducible domain (KID) of the transcription factor CREB (cyclic AMP-responsive element binding protein) exhibit a decrease in fluorescence resonance energy transfer upon phosphorylation of the KID by the cyclic AMP-dependent protein kinase, PKA (Y. Nagai et al., 2000, A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo, Nature Biotech. 18: 313-316). Calmodulin, a calcium-sensitive protein, has been inserted into YFP, resulting in calcium sensors (‘camgaroos’) that increase fluorescence sevenfold upon binding of calcium (G S Baird et al., 1999, Circular permutation and receptor insertion within green fluorescent proteins, Proc. Natl. Acad. Sci. USA 96: 11241-11246). Similarly, insertion of a circularly permuted GFP between calmodulin and M13—a peptide that binds calmodulin in a calcium-sensitive manner—yields calcium indicators that are known as ‘pericams’ (T. Nagai et al., 2001, Circularly permuted green fluorescent proteins engineered to sense Ca2+, Proc. Natl. Acad. Sci USA 98: 3197-3202). Alternative calcium indicators known as ‘cameleons’ have been created by sandwiching calmodulin, a peptide linker, and M13 between CFP and YFP (A. Miyawaki et al., 1997, Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin, Nature 388: 882-887).
Transcriptional reporter assays provide a measurement of pathway activation/inhibition in response to an agonist/antagonist and have been used extensively in GPCR studies (see Klein et al., U.S. Pat. No. 6,255,059 and references therein). Reporter assays couple the biological activity of a receptor to the expression of a readily detected enzyme or protein reporter. Synthetic repeats of a particular response element can be inserted upstream of the reporter gene to regulate its expression in response to signaling molecules generated by activation of a specific pathway in a live cell. Such drug screening systems have been developed with a variety of enzymatic and fluorescent reporters, including β-galactosidase (H Brauner-Osborne & M R Brann, 1996, Eur. J. Pharmacol. 295: 93-102), luciferase, alkaline phosphatase, GFP, β-lactamase (G. Zlokarnik et al., 1998, Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter, Science 279: 84-88) and other reporters. Transcription reporter assays are highly sensitive screening tools; however, they do not provide information on the mechanism of action of the compound, enable mapping of the components of the pathway leading to transcription, or enable studies of individual steps within signaling cascades.
Subcellular compartmentalization of signaling molecules is an important phenomenon in cell signaling, not only in defining how a biochemical pathway is activated but also in influencing the desired physiological consequence of pathway activation. High-content screening (HCS) is an approach that relies upon imaging of cells to detect the subcellular location and trafficking of proteins in response to stimuli or inhibitors of cellular processes. Fluorescent probes can be used in HCS. For example, GTP has been labeled with the fluorescent dye, BODIPY, and used to study the on and off-rates of GTP hydrolysis by G-proteins, and fluorescein-labeled myristoylated Galpha-I has been used as the ligand bound to Gbeta-gamma in order to study the association and dissociation of G-protein subunits (N A Sarvazyan et al., 2002, Fluorescence analysis of receptor-G protein interactions in cell membranes, Biochemistry 41: 12858-12867).
Increasingly, green fluorescent protein (GFP) has been used to analyze key signaling events within cells. By fusing in-frame a cDNA for GFP to a cDNA coding for a protein of interest, it is possible to examine the function and fate of the resulting chimera in living cells. This strategy has now been applied to nearly all known elements of G-protein coupled pathways including the receptors themselves; G-protein subunits such as Gα; beta-arrestin; RGS proteins; protein kinase C; and numerous other intracellular components of G-protein-coupled pathways (M Zaccolo and T. Pozzan, 2000, Imaging signal transduction in living cells with GFP-based probes, IUBMB Life 49: 1-5, 2000.)
For example, G-protein-coupled receptors have been tagged with GFP in order to monitor receptor internalization. A fusion protein comprising GFP-beta-arrestin has been shown to co-localize with thyrotropin-releasing hormone receptor 1 in response to agonist (T Drmota et al., 1999, Visualization of distinct patterns of subcellular redistribution of the thyrotropin-releasing hormone receptor-1 and Gqalpha/G11alpha induced by agonist stimulation, Biochem. J. 340: 529-538). GFP has been introduced internally to G-proteins, creating a Galpha/GFP chimera, which has been shown to translocate to the cell membrane upon GPCR activation (J-Z Yu & M Rasenick, 2002, Real-time visualization of a fluorescent GalphaS dissociation of the activated G protein from plasma membrane, Mol. Pharmacol. 61: 352-359; P Coward et al., 1999, Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors, Anal. Biochem. 270: 242-248). GFP tagging has also been used to monitor intracellular signaling events. GFP-tagged Regulator of G protein Signaling (RGS2 and RGS4) proteins were selectively recruited to the plasma membrane by G proteins and their cognate receptors (AA Roy et al., 2003, Recruitment of RGS2 and RGS4 to the plasma membrane by G proteins and receptors reflects functional interactions. Mol. Pharmacol. 64: 587-593). GFP-tagged protein kinase C (PKC), which is activated by release of diacylglycerol from cell membranes, has been used to monitor translocation of the kinase in response to cell signaling (E. Oancea et al., 1998, Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells, J. Cell Biol. 140: 485-498). GFP-tagged connexin has been used to monitor intracellular calcium flux (K Paemeleire et al., 2000, Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin, Mol. Biol. Cell 11: 1815-1827). GFP-tagged beta-arrestin has been used to monitor GPCR activation by imaging the subcellular redistribution of beta-arrestin in reponse to GPCR agonist. The latter assay, known as TransFluor, is marketed by Norak Bioscience (www.norakbio.com) and is the subject of U.S. Pat. Nos. 5,891,646 and 6,110,693. All the above assays and inventions involve fusing a protein of interest (receptor, beta-arrestin, G-protein, connexin, RGS, kinase etc.) to an optically detectable molecule such as GFP; expressing the fusion construct in cells; and then detecting the quantity, and/or the subcellular location, of the chimeric protein in response to a stimulus or inhibitor.
Measurements of protein-protein interactions between GPCRs and cognate intracellular signaling proteins represent an alternative to the above-mentioned techniques. In contrast to monitoring a single protein by tagging it with GFP, a protein-protein interaction assay is capable of measuring the dynamic association and dissociation of two proteins. The most widespread cell-based assays for protein-protein interactions are based on fluorescence resonance energy transfer (FRET) or bioluminescence resonance energy transfer (BRET). With FRET, the genes for two different fluorescent protein reporters are separately fused to genes encoding of interest, and the two chimeric proteins are co-expressed in live cells. When a protein complex forms between two proteins of interest, the two fluorophores are brought into close proximity. If the two proteins possess overlapping emission and excitation wavelengths, the emission of photons by the first “donor” fluorophore, results in the efficient absorption of the emitted photons by the second “acceptor” fluorophore. The FRET pair fluoresces with a unique combination of excitation and emission wavelengths that can be distinguished from those of either fluorophore alone in living cells. Quantifying FRET or BRET can be technically challenging its use in imaging protein-protein interactions is limited by the very weak FRET signal. The signal is often weak because the acceptor fluorophore is excited only indirectly, through excitation of the donor. The fluorescence wavelengths of the donor and acceptor must be quite close for FRET to work, because FRET requires overlap of the donor emission and acceptor excitation. Newer methods are in development to enable deconvolution of FRET from bleed-through and from autofluorescence. In addition, fluorescence lifetime imaging microscopy eliminates many of the artifacts associates with quantifying simple FRET intensity.
For example, FRET has been used to study GPCR-mediated activation of G-proteins in living cells (C. Janetopoulos, 2001, Receptor-mediated activation of heterotrimeric G-proteins in living cells, Science 291:2408-2411) and to study the association of PKA with AKAPs (M L Ruehr et al., 1999, Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins, J. Biol. Chem. 274: 33092-33096). A variety of GFP variants, including cyan, citrine, enhanced green and enhanced blue fluorescent proteins, have been used to construct FRET assays. With BRET, a luminescent protein, such as the enzyme Renilla luciferase (Rluc) is used as the energy donor and a green fluorescent protein (GFP) is used as the acceptor. Upon addition of a compound that serves as the substrate for Rluc, the FRET signal is measured by comparing the amount of blue light emitted by Rluc to the amount of green light emitted by GFP. The ratio green/blue ratio increases as the two proteins are brought into proximity. FRET and BRET have been applied to studies of GPCR oligomerization for oligomers of the β2-adrenergic, δ-opioid, thyrotropin releasing hormone and melatonin receptors. BRET has also been used for studies of the agonist-dependent association of beta2-arrestin with the beta2-adrenergic receptor in live cells (S Angers et al., 2000, Detection of beta-2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer, Proc. Natl. Acad. Sci. USA 97: 3684-3689). Receptor ligands, coupled to fluorophores, have also been used as FRET partners to monitor oligomerization of GPCRs.
In principle, cell-based assays of protein-protein interactions can be used both to monitor the activity of a biochemical pathway in the living cell and to directly study the effects of chemicals on targets and pathways. Unlike transcriptional reporter assays, the information obtained from perturbation of a specific pathway is what is happing specifically in a particular branch or node of that pathway, not its endpoint. Protein-fragment complementation assays (PCAs) and enzyme-fragment complementation assays represent an alternative to FRET-based methods. PCA involves tagging of proteins with polypeptide fragments derived by fragmenting a suitable reporter. Unlike intact fluorescent proteins or holoenzymes, the PCA fragments have no intrinsic activity or fluorescence. However, if two proteins that are tagged with complementary fragments interact, the fragments are brought into close proximity. The complementary fragments can then fold into an active conformation and re-constitute the activity of the reporter from which the fragments were derived. Unlike FRET or BRET, PCA-based fluorescent or luminescent assays provide for signals with large dynamic range. Moreover, PCAs do not require specialized optics or equipment. Using a similar approach, naturally-occurring subunits of a multimeric protein—beta-galactosidase—have been used to construct complementation assays for the measurement of protein-protein interactions (Rossi, et al., 1997, Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci USA 94: 8405-8410).
Fluorescent PCAs based either on dihydrofolate reductase or beta-lactamase have been used to quantify the effects of the drug rapamycin on its target in living cells (Remy, I. and Michnick, S. W., Clonal Selection and In Vivo Quantitation of Protein Interactions with Protein Fragment Complementation Assays. Proc Natl Acad Sci USA, 96: 5394-5399, 1999; Galameau, A., Primeau, M., Trudeau, L.-E. and Michnick, S. W., A Protein fragment Complementation Assay based on TEM1 β-lactamase for detection of protein-protein interactions, Nature Biotech. 20: 619-622, 2002 and to study phosphorylation-dependent interactions of two domains of the cyclic AMP response element binding protein, CREB (J M Spotts, R E Dolmetsch, & M E Greenberg, 2002, Time-lapse imaging of a dynamic phosphorylation-dependent protein-protein interaction in mammalian cells, Proc. Natl. Acad. Sci. USA 99: 15142-15147.) PCA has also been used to construct quantitative and high-content assays for a variety of proteins in the insulin and growth factor-dependent pathways in mammalian cells (Remy, I. and Michnick, S. W., Visualization of Biochemical Networks in Living Cells, Proc Natl Acad Sci USA, 98: 7678-7683, 2001 and U.S. Patent Application 20030108869).
With regard to direct assays of receptor activation, PCA has been used to construct fluorescent assays of the erythropoietin (EPO) receptor in living cells (Remy, I., Wilson, I. A. and Michnick, S. W., Erythropoietin receptor activation by a ligand-induced conformation change, Science 283: 990-993, 1999; Remy, I. and Michnick, S. W., Clonal selection and in vivo quantitation of protein interactions with protein fragment complementation assays, Proc Natl Acad Sci USA, 96: 5394-5399, 1999; and U.S. Pat. No. 6,294,330). These assays were quantitative, demonstrating dose dependence and showing a differential response to erythropoietin or EMP1 consistent with the EC50 of the two agonists. Similarly, enzyme-fragment complementation assays based on low-affinity subunits of β-galactosidase have been used to study EGF receptor dimerization in living cells (Rossi, et al., Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation, Proc Natl Acad Sci USA 94: 8405-8410, 1997; and U.S. Pat. No. 6,342,345). However, the prior art is silent on the use of either protein-fragment or enzyme-fragment complementation assays for G-protein-coupled receptors or G-protein-coupled signaling pathways.
At its basic level, fragment complementation is a general and flexible strategy that allows measurement of the association and dissociation of protein-protein complexes in intact, living cells. In particular, PCA has unique features that make it an important tool in drug discovery:                Molecular interactions are detected directly, not through secondary events such as transcription activation or calcium release.        Tagging of proteins with large molecules, such as intact, fluorescent proteins, is not required.        With in vivo PCAs, proteins are expressed in the relevant cellular context, reflecting the native state of the protein with the correct post-translational modifications and in the presence of intrinsic cellular proteins that are necessary, directly or indirectly, in controlling the protein-protein interactions that are being measured by the PCA.        PCA allows a variety of reporters to be used, enabling assay design specific for any instrument platform, automation setup, cell type, and desired assay format. Reporters suitable for PCA include fluorescent, phosphorescent and luminescent proteins (GFP, YFP, CFP, BFP, RFP and variants thereof, and photoproteins (aequorin or obelin); various luciferases; β-lactamase; dihydrofolate reductase; beta-galactosidase; tyrosinase; and a wide range of other enzymes.        Depending upon the choice of reporter, either high-content or high-throughput assays can be constructed with PCA, allowing flexibility in assay design depending on the specific target and the way in which it responds to agonist or antagonist in the cellular context.        With high-content PCAs, the sub-cellular location of protein-protein complexes can be determined, whether in the membrane, cytoplasm, nucleus or other subcellular compartment; and the movement of protein-protein complexes can be visualized in response to a stimulus or inhibitor.        With high-throughput PCAs, the assays are quantitative and can be performed either by flow cytometry or in multi-well, microtiter plates using standard fluorescence microplate readers.        PCA can be used to ‘map’ proteins into signaling pathways and validate novel targets by detecting the interactions that a particular protein makes with other proteins in the context of a mammalian cell, and then determining whether the protein-protein complex can be modulated in response to an agonist, antagonist or inhibitor.        